Selection of single cells producing a therapeutic glycoprotein with desired property using a microwell array

Abstract

Mammalian cells are commonly used for the production of antibodies and other therapeutic proteins. Mammalian cell lines are chosen for their post translational modification (PTM) ability, which makes produce proteins more effective and having fewer side effects. Genetically engineered cell lines used in the production have population heterogeneity in their growth rate, protein production yield and the protein’s glycan profile, even though the population is originated from a single parental cell. This heterogeneity presents a barrier to the enhancement of protein yield and protein’s quality. Due to the continuously increasing demand for recombinant products such as monoclonal antibodies, specific productivity still needs to be optimized. And the therapeutic efficacy and in vivo biological function of a glycoprotein is significantly affected by its glycosylation profile. For the development of glycoproteins with thera-peutic applications, selection of cell lines producing a glycoprotein with adequate glycoform is crucial. Here we demonstrate an array-based analysis of secreted proteins for rapid and efficient selection of a single cell producing an antibody with high productivity and a glycoprotein with desirable glycosylation. Our approach relies on microengraving and interrogation of therapeutic proteins produced by individual cells in a microwell array in terms of glycosylation profile as well as the produced amount. Based on statistical analysis of the interrogation, corresponding single cell is selected, retrieved, and expanded. We applied the approach to human immunoglobulin G (hIgG) for selection of high productive cells. And we also applied to human recombinant erythropoietin (rhEPO)-producing CHO cells for selection of a single CHO cell that produces rhEPO with a high sialylation degree. This method can find widespread use in the clonal selection for the production of other therapeutic proteins with specific glycosylation as well as high productivity in ...